One process for discovering new therapeutically active compounds for a given indication involves the screening of all compounds from available compound collections. From the compounds tested, one or more structures is selected as a promising lead. A large number of related analogues are then synthesized in order to develop a structure-activity relationship (SAR). The SARs direct the development and then selection of one or more optimal compounds following traditional one-at-a-time synthesis and biological testing. This optimization process is long and labor intensive.
Adding significant numbers of new structures to the compound collections used in this initial screening step of the discovery and optimization process cannot be accomplished with traditional one-at-a-time synthesis methods, except over a time frame of months or even years. Faster methods are needed that allow for the preparation of libraries of related compounds in a matter of days or a few weeks. This need is particularly apparent when it comes to synthesizing more complex compounds.
Combinatorial approaches have recently been extended to “organic” or non-peptide, libraries. The organic libraries at present, however, are limited in diversity and generally relate to peptidomimetic compounds; in other words, organic molecules that repeat a peptide chain pharmacophore. There is a need in the art for additional approaches to the preparation of new organic libraries.
Cytokines are pleiotropic extracellular proteins that are released and recognized by a wide variety of cell types. Via a series of complex interactions they are responsible for regulating many of the events involved in growth and differentiation of an organism. Among the cytokines, tumor necrosis factor-α (TNF-α) has been shown to play an important role in the genesis of certain chronic inflammatory and autoimmune diseases. TNF-α is secreted mainly by macrophages and monocytes in response to a variety of inflammatory agents. Other cell types such as NK cells, T cells, B cells, Kupfer cells, and glial cells also produce TNF-α.
TNF-α is synthesized as an inactive 26 kDa pro-protein which is cleaved by the metalloprotease TNF-α Converting Enzyme (TACE) to afford the active 17 kDa cytokine protein. The cytokine exerts its biological effects by interacting with two high affinity receptors of molecular weights 55 kDa (TNFR1 or p55) and 75 kDa (TNFR2 or p75) found on the surface of most cell types. As a result of TNF-α binding to its receptors, a cascade of signaling events occurs within the cell. The exact nature and sequence of events is dependent upon cell type and receptor. Two of the most important physiological effects of TNF-α binding to its receptors are the upregulation of new genes by activation of the transcription factor NFκB, and induction of programmed cell death or apoptosis.
Apoptosis is a normal physiological process whereby incompetent cells become targeted for disposal by the immune system. The process involves a series of morphological changes to the apoptotic cell, including a change of surface chemistry. This change in surface chemistry is recognized by macrophages that rapidly phagocytose the cell. A number of stimuli can induce apoptosis, including DNA damage, UV radiation, growth factor deprivation, bacterial and viral infection, and ligation of cell surface receptors. TNF-α has been shown to induce apoptosis by binding to TNFR1. Under normal biochemical circumstances the process of apoptosis is integral in regulating the homeostatic balance between cell death and cell proliferation. However in many autoimmune diseases this balance is shifted; not only do unwanted cells undergo apoptosis but healthy cells as well. These diseases are often associated with increased levels of TNF-α. There is a need in the art for compounds that can modulate binding of TNF-α to cell receptors, and/or modulate the consequential intracellular events.
Transcription factors are a family of proteins that bind to DNA and serve to upregulate gene expression. Often they remain in an inactive form until acted upon by a biochemical signal. One such transcription factor is nuclear factor kappa B (NFκB), which can be activated by the binding of TNF-α to TNFR1 and/or TNFR2. NFκB regulates many of the cytokines and other proinflammatory molecules associated with inflammatory and autoimmune diseases. Classes of proteins subject to regulation by NFκB and which have been demonstrated to be involved with disease states are cytokines and growth factors, adhesion molecules, chemokines, and immunoreceptors.
The inhibition of TNF-α induced apoptosis and of NFκB activation is one means of preventing and/or treating autoimmune and inflammatory diseases including, but not limited to, rheumatoid arthritis, inflammatory bowel disease, psoriasis, atherosclerosis, asthma, reperfusion injury, ischemia, sepsis, graft vs. host disease, adult respiratory distress syndrome, multiple sclerosis, and a host of severe invasive infections such as fulminant hepatitis, AIDS and bacterial meningitis, and allergic inflammation of the lungs and airways.
Interleukin-8 (IL-8) is a chemokine (chemotactic cytokine) which plays an important role in the recruitment of neutrophils to sites of inflammation. It is a member of the CXC subfamily of chemokines, members of which contain a single amino acid residue between the first two cysteines. In addition to inducing the chemotaxis of neutrophils, IL-8 exerts other immunomodulatory effects such as release of superoxide, mobilization of intracellular Ca++, upregulation of cell surface integrins, and activation of phospholipase D. All of these cellular events are the result of IL-8 binding to one of its two high affinity receptors. The two receptors, known as IL8RA or CXCR1 and IL8RB or CXCR2, bind the ligand with a Kd of ca. 2 nM.
Numerous reports in the literature have associated increased levels of IL-8 with the development of inflammatory and autoimmune diseases such as Inflammatory Bowel Disease (IBD), psoriasis, rheumatoid arthritis, Acute Respiratory Distress Syndrome (ARDS), cancer, atherosclerosis, reperfusion injury, and graft vs. host disease. The inhibition of IL-8 or other CXC chemokines from binding to CXCR1 and/or CXCR2 receptors is one means of preventing and/or treating these diseases.
Although treatment regimens are available for the symptomatic amelioration of some or all of these diseases, there still exists the need for compositions and methods for preventing and/or treating the inflammation which is often associated with the disease.
The present invention satisfies these needs and provides related advantages as well, as described more fully herein.